Method of making complex nano particles and using the same to reduce cell viability

ABSTRACT

The development of anticancer metal-based drugs was done by reacting oyelamine with selenous acid to produce a quaternary ammonium salt which consequently converted to platinum and cobalt cationic complexes via complexing the first compounds with platinum (II) or cobalt (II) ions. The surface properties studies that were conducted included critical micelle concentration (CMC), maximum surface excess (Γmax) and minimum surface area (Amin). Free energy of micellization (ΔG° mic) and adsorption (ΔG° ads) were calculated. Antitumor activities were tested by using Ehrlich Acites Carcinoma (EAC) as a model system of mice cell tumor. These compounds were also tested in vitro on human five monolayer tumor cell lines: MCF7 (Breast carcinoma), HEPG2 (liver carcinoma), and HCT116 (colon carcinoma), etc. FTIR spectra, elemental analysis and H1 NMR spectrum were performed to insure the purity of the prepared compounds.

FIELD OF TECHNOLOGY

This disclosure relates generally to a method of making a complex nanoparticle and using the same to reduce cell viability. More specificallyCobalt and/or Platinum are used with oyelammonium hydrogen selenite toform a complex that would be used to reduce tumor size or bacterialgrowth by reducing cell viability.

BACKGROUND

Cationic surfactants have been the focus of wide spread interest overdecades due to their ability to self-assemble in super molecularstructures such as micelles. The aggregates formed create sharp polaritygradients at the interface and define clear hydrophobic regions in anaqueous solution. Those properties are of fundamental importance for thecreation of new materials.

Surfactant metal complexes are expected to provide a wide range ofinteresting phenomena on the aggregation behavior in aqueous solutiondue to a variety of their charge numbers, size and extent ofhydrophopicity by a combination of a central metal and ligands. However,their physical properties in solutions have not been extensivelystudied. In the studies so far performed, novel characters of surfactantmetal complexes have been revealed, and the results should providesignificant information on surfactant solution chemistry.Metaloaggregates are made of surfactants that combine ametal-coordinating polar head to hydrophobic tail. The polar head of thesurfactant is functionalized with metal ions are bound to and surroundedby hydrophobic region, similar to the situation found inmetalloproteinase. The antimicrobial action of cationic surfactant isbased on their ability to disrupt the integral bacterial membrane by acombined hydrophobic and electrostatic adsorption phenomenon at themembrane water interface making disorganization. The pathogenicbacterial cell membrane is predominantly negatively charged as comparedwith eukaryotic cells. Hence the positive charge of the cationicamphiphile facilitates their interactions with bacterial membrane.

The main goal of cancer therapy is to attain maximum therapeutic damageof tumor cells in combination with minimum concentration of the drug.This can be achieved in principle via selective antitumor preparations,the cytostatic effects of which would be restricted within tumor tissue.While 100% selectivity may be impractical, achievement of reasonablyhigh selectivity seems to be a feasible aim. The bioenergetic status intumor was selective and affected by the Metal complexes. Minimization ofsignals of high-energy phosphate was observed after injection of thecomplexes. An increase of the number of DNA single-strand breaksregistered in tumor tissue, supporting the suggestion that complexes maydirectly affect DNA, but the action of these complexes as antitumoragents found to be dependent on the type of tumer cell line tested.

SUMMARY

The invention discloses a method of making and using a complex nanoparticle as antitumor agent. More specifically Cobalt and/or Platinumare used with olylammonium hydrogen selenite as complex to reduce cellviability.

In one embodiment, a method of making the olylammonium hydrogen seleniteis described. In another embodiment, a method of making the complex ofolylammonium hydrogen selenite and Cobalt (Co) is described. In anotherembodiment, a method of making the complex of olylammonium hydrogenselenite and Platinum (Pt) is described.

In one embodiment, nanoparticle comprising of cyclodextrin and Co/Ptcomplex is described. In another embodiment, characterization of thenanoparticle (cyclodextrin and Co/Pt complex) is performed to provesuperior functional qualities of the nanoparticle.

In one embodiment, cancer cell lines of various concentration and typeswere treated with nanoparticle comprising of cyclodextrin and Co/Ptcomplex and antitumor activity were observed. In one embodiment, growthof bacteria is reduced by reducing the cell viability using nanoparticlecomprising of cyclodextrin and Co/Pt complex.

Other features will be apparent from the accompanying drawings and fromthe detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Example embodiments are illustrated by way of example and not limitationin the figures of the accompanying drawings, in which like referencesindicate similar elements and in which:

FIG. 1 shows the picture of nanoparticle comprising of cyclodextrinandolylammonium hydrogen selenite Co complex using Transmission ElectronMicroscopy (TEM).

Other features of the present embodiments will be apparent from theaccompanying the detailed description that follows.

DETAILED DESCRIPTION

In this invention a method to make the nanoparticle comprising ofolylammonium hydrogen selenite and Co/Pt complex is discussed. Themethod of using the nanoparticle comprising of olylammonium hydrogenselenite and Co/Pt complex for inhibition of cell viability in mammaliancells as well as bacterial cell are disclosed.

Synthesis of Olylammonium Hydrogen Selenite:

All chemicals used here were bought from Sigma Aldrich USA. One mole ofselenous acid was mixed with one mole of oyelamine at room temperaturein ethyl alcohol to make a mixture. The mixture was stirred until theformation of precipitate slowed down or stopped. One can visuallyascertain the slowing of precipitate formation or stopping of theprecipitate formation due to color formation of the precipitate. The(white yellowish, reddish and pink) precipitate (precipitate 1) wasfiltered and washed by ethyl alcohol, then recrystallized by addingdiethyl ether (precipitate 2).(¹⁷) The precipitate 2 (designated asII_(a)) has the general formula as follows:RN⁺H₃HSeO₃ (where R=olyl)  (Eq 1)

Synthesis of metal complexes: Synthesis of cobalt (II) or platinumhydrogen selenite dehydrate is performed by adding selenius acid(H2SeO3) with basic cobalt (II) carbonate (Co (OH2)2 CO3) or platinumcarbonate (Pt (OH2) 2 CO3). Basic cobalt (II) carbonate (Co (OH2)2 CO3)or platinum carbonate (Pt (OH2) 2 CO3) solutions were prepared by mixingequimolar amounts of CoCl2 or PtCl2 (cobalt or platinum chloride) andNa2CO3 (sodium carbonate). The solid precipitate of Cobalt carbonate(precipitate 3) was washed till the absence of foreign ions.2H₂O+CoCl₂+Na₂CO₃→CoCO₃.2H₂O+2NaCl  (Eq 2)

An aqueous solution of H₂SeO₃, 2 g in 10 ml water (0.016 mol) was addedto a warm solution of the freshly prepared Cobalt (Co) carbonate, 1.22 gin 10 ml water (0.008 mol). The Co carbonate and H₂SeO₃ mixed solutionis then filtered and kept at room temperature for crystallization. Itwas observed that after 2 days crystalline prisms of red color crystalswere formed. The crystals were first filtered, washed with water anddried in air.(¹⁸) For obtaining platinum (II) hydrogen selenitedihydrate an aqueous solution of 2 g H₂SeO₃ in 10 ml water was added toa warm solution of the freshly prepared Pt carbonate (precipitate 4)1.28 g in 10 ml water. The obtained solution is filtered and kept atroom temperature for crystallization after 24 hour; crystalline prismsof blue color are formed. The temperature of removal of 2 molecules ofwater is 100-110° C.

Synthesis of Cobalt or Platinum Fatty Olylammonium Hydrogen SeleniteComplexes:

Cobalt or platinum fatty olylammonium hydrogen selenite complexes wereprepared by refluxing two moles of olylammonium hydrogen selenites(II_(a)) with one mole of cobalt or platinum hydrogen selenite in ethylalcohol for two hours. The product 1 or 2 is designated as (IIb andIIc).2RN⁺H₃(HSeO₃)⁻+M(HSeO₃)₂→[RNH3]⁺ ₂M[HSeO₃]⁻ ₄  (eq 4)

The product 1 or 2 is purified and recrystallized three times inpetroleum ether and then washed with diethyl ether. The products kept indesiccators till used. General formula for the metal complexes may beshown as below:[RN⁺H₃]₂[M(HSeO₃)₄]⁻² (Where R=oyelamine and, M: CO⁺² or Pt⁺²).  (Eq. 5)

Method of Making the Nanoparticle Comprising of Cyclodextrin andOlylammonium Hydrogen Selenite Co/Pt Complex:

The Co or Pt complex was mixed mechanically very well with thecyclodextrin oligosaccharide using vortex then both are ground to thenano sized particles using ball mill model PM 400 at 200 rpm for 10hours. Planetary Ball Mills are used wherever the highest degree offinesse is required. Apart from the classical mixing and size reductionprocesses, the mills also meet all the technical requirements forcolloidal grinding and have the energy input necessary for mechanicalalloying processes. The extremely high centrifugal forces of thePlanetary Ball Mills result in very high pulverization energy andtherefore short grinding times. The product loaded cyclodextrin complexnanoparticles was obtained and their particle size was determined usingtransmission electron microscope.

FIG. 1 shows the nanoparticle comprising of cyclodextrin and Co complex.The compound is oyelamine hydrogen selenite cobalt complex but forsimple form we can say Co complex, then we comprising it withcyclodextrin (In the pharmaceutical industry, cyclodextrins have mainlybeen used as compressing agents to increase the grinding ability and theaqueous solubility of poorly water-soluble drugs, and to increase theirbioavailability and stability. In addition, cyclodextrins can be used toreduce gastrointestinal and ocular irritation, reduce or eliminateunpleasant smells or tastes, prevent drug-drug or drug-additiveinteractions, or to convert oils and liquid drugs into microcrystallineor amorphous powders.

TABLE 1 The critical micelle concentration (CMC) and surface parameterammonium hydrogen selenite surfactants CMC × γCMC ΠCMC PC20 Γmax × 10−11A min Δ Gads/ Comp. 10−3 (mN/m) (mN/m) (Mole/L) (Mole/cm²) (nm2) Δ GadsΔ Gmic Amin IIa 1.3 33 39 3.8 10.6 1.6 −67.7 −34.1 −46.8 IIb 1.1 32 404.0 10.8 1.5 −69.9 −34.8 −49.1 IIc 0.70 30 42 4.1 11.3 1.45 −71.1 −35.3−50.2

As shown from previous table by complexing parent surfactants withcobalt or platinum ions, high depression was observed in CMC values.That fact could be explained from the unique property of the metalcomplexes in water. That is the complexes retain its unity in theirsolutions, which increased their volume in the aqueous media and thenrepulsion is occurred between the hydrophobic chain and water molecules.(IIc) was found to be the most efficient one in because it achieved themaximum reduction of the surface tension at CMC. The efficiency “PC20”increase with increasing molar ratio of methylene units. These due tothe fact that the efficiency of adsorption at interfaces increaselinearly with increase in the carbon atoms in hydrophobic group. In caseof the prepared parent cationic surfactants by increasing the numbermethylene units Maximum surface excess Γmax increases, this due tomigration of molecules to the water-air interface. The consequenceincrease of Γmax leads to crowdiness occurred at the interface whichcausing decrease in Minimum area per molecule Amin values. That is dueto the minimum surface area decrease with increasing the hydrophobicchain length of the synthesized surfactant molecules. The standard freeenergies of micellization ΔG° mic and adsorption ΔG° ads values arealways negative indicating the spontaneously of these two processes butthere is more increase in negativity of ΔG° ads rather than those ofmicellization indicating the tendency of the molecules to be adsorbed atthe interface.

Antitumor Action of the Prepared Compounds:

Olylammonium hydrogen selenites hydrogen selenite with its cobalt andplatinum complexes were investigated as potential selective, anticancerprodrugs. They were tested by using Ehrlich Acites Carcinoma (EAC) as amodel system of mice cell tumor. These compounds were also Tested invitro on human five monolayer tumor cell lines: MCF7 (Breast carcinoma),HEPG2 (liver carcenoma), U 251 (Hela tumor) and HCT116 (coloncarcinoma).

Evaluation of Antitumor Activity of the (EAC):

Ehrlich ascites carcinoma cells as a model system was based on thefinding that it is excellent tool for studying the biological behaviorof malignant tumors and drug action with cells. A line of Ehrlichascites carcinoma (EAC) which used in the present study had been kindlysupplied from National Cancer Institute, Cairo, Egypt, and maintained infemale Swiss albino mice through weekly IMP transplantation of 2.5×106tumor cells/mouse. EAC cells were obtained by needle aspiration withaseptic condition. The ascetic fluid was diluted with sterile saline sothat 0.1 ml contains 2.5×106 cells counted microscopically using ahaemocytometer. In vitro studying of these compounds antitumor activitywas determined according to the percentage of nonviable cells (NVC %)which was calculated by the following equation NVC %=[number ofNVC/total number of cells]/100.

TABLE 2 Antitumor activity of the prepared compounds using (EAC): %Inhibition of cell viability Sample μg/ml Conc. μg/ml 100 50 25 IIa 40%20% 10% IIb 100% 80% 40% IIc 90% 70% 30%

As shown from Table 2 increasing the concentration of olylammoniumhydrogen selenite (IIa) and its cobalt (IIb) or platinum (IIc) complexesin the EAC media was accompanied by progressive increase in the percentof non-viable cells. This comes from the fact that by increasing theconcentration of cationic surfactant the adsorption of ions on cellmembrane increases, leading to increase in penetration and antitumoractivity. The inhibition of cell viability percent showed that the IIb(cobalt complex) is the most active one at concentration 100 μg/ml, thepercentage of non-viable cell reach to 100%. This mean that the drug atthis concentration cause death all the tumor cell while, atconcentration 50 μg/ml the percentage of reach to 80%. But, atconcentration 25 μg/ml the (NVC %) reach to 40%. While IIc (platinumcomplex) at concentration 100-μg/ml (NVC %) reach to 90% and atconcentration 50 μg/ml reach 70%. From these results IIa,c are the mostactive of all derivatives, since cobalt complexes seem to offer promisedue to high electron affinity of the metal which increasing the abilityto bind DNA and the ready reducibility of the compounds. While, it canbe seen that IIa has the least toxic effect of all derivatives on EACcells.

Evaluation of Cytotoxic Activity on Human Tumor Cell Lines:

The results of the cytotoxic activity on human tumor cell lines weredetermined according to the dose values of drug exposure for cell linesto reduce survival to 50% (Ic50). The experimental results recorded inTable (3).

TABLE (3) Cytotoxic activity of the IIb,c compounds on human cell lineCell lines HELA Sample (IC50) MCF7 (IC50) HEPG2 (IC50) HCT116 (IC50) IIb2.08 o.6 0.94 −Ve IIc −Ve 0.47 1.41 −Ve

The compounds tested exhibited high activity in vitro system on thetumor cell line investigated, IIb have the highest cytotoxic effect onMCF7, HEPG2 and HELA. The dose of it at which the survival reduction to50% is (Ic50=0.6, 0.94 and 2.8 μg/ml), respectively. Also IIc show goodcytotoxic activity on HEPG2 and MCF7 (Ic50=1.41 and 0.47 μg/ml),respectively. It should be noted that the action of these compounds asantitumor agents found to be dependent on the type of tumor cell linetested, but as shown from the results IIb (cobalt complexes) showexcellent cytotoxic activity against several tumor cell line and undervery low concentration reduces the survival to 50%. This comes from thefact that cobalt complexes have a capacity to reduce the energy statusin tumors as well as enhance the tumor hypoxia which also influencestheir antitumor activities. It may be also concluded that the level ofcellular damage inflicted by these complexes depends on the nature oftheir axial ligands. There is evidence that cobalt complexes causesignificant changes in metabolism namely activation of lipidperoxidation, DNA damage and reduction of the bioenergetic status oftumor tissues. In general high selectivity of action by redox—activecobalt complexes upon tumors is due to their specific reactivity.Platinum complexes exhibit superoxide dismutase like activity which usedas anti-inflammatory agent and lipid soluble. This property enables thecompound to penetrate membranes and become inter-cellular. Finallyplatinum and cobalt complex surfactant nanoparticles in our researchaffect tumor tissue at very low concentration at values lower than theirCMC values, which mean that there is a strong relation between verysmall values of CMC of these compounds and the reaching to Ic50 valuesunder very low concentration, this due to the fact that increasingconcentration of cationic surfactant causes increase the adsorptionprocess on cell membrane till reaching the CMC, after this concentrationthe adsorption retarded slowly then stopped due to form micelles whichprevent the mobility and suppress antitumor activity. Oyelammoniumhydrogen selenite does not reach to Ic50 for all tested human monolayertumor cell lines. Many targets may be explored to counteract cancer andindication the role of studied metals should be useful for a better useof metal-based anticancer drugs.

Antibacterial Activity of the Prepared Surfactants Against SulphurReducing Bacteria:

Sulphur reducing bacteria are mainly sulfate reducers, and their growthfrequently causes severe corrosion problems in oil well pipes. Due tothe economic losses as well as environmental health and safety hazardscaused by the activity of stabilized mixed culture containing sulphatereducing bacteria, (SMC-SRB) in many industrial sectors such as the oiland gas industry, it was important to minimize the risks resulting fromSRB activity.

Sulfur reducing bacteria are strict anaerobes that are often found inbiotopes where toxic conditions can temporarily existing. The bacteriahave developed several defense strategies in order to survive exposureto oxygen. These strategies include peculiar behaviors in the presenceof oxygen, like aggregation or aerotaxis, and enzymatic systemsdedicated to the reduction and the elimination of oxygen and itsreactive species.

Quaternary ammonium compounds are most effective against anaerobicbacteria (e.g. those that occur in oil wells). Several studies indicatedthat some quaternary ammonium compounds act as corrosion inhibitors anddecrease sulfide production by (SRB) at low concentration than somebiocides of commercial source. The results of the synthesized cationicsurfactants against sulphur reducing bacteria recorded in Table 4.

TABLE 4 Inhibition zone diameter (mm/mg sample) for the synthesizedcationic surfactants against sulphur reducing bacteria. Inhibition zonediameter (mm/mg sample) Sulphur Sample reducing bacteria IIa 25 IIb 24IIc 23

The results in Table 4 indicates that the new synthesized cationicsurfactants have high antimicrobial activity against sulphur reducingbacteria, and the difference in activity depends on the length ofhydrophobic chain. The optimal length of the alkyl chain has been notedto be ten carbon atoms. The highest results were achieved by platinumcomplexes, this may be due to platinum is oxidizing agent act asreduction inhibitors leading to decrease in sulfide production anddecreasing the growth rate of anaerobic (SRB). In more general bacterialgrowth Inhibition by metal ions was investigated in the sulphate-freemedium. The rate of H₂S production was approximately directlyproportional to the specific activities of the invested enzymes. Theseactivities were inversely proportional to the generation time. The rateof microbiologically induced corrosion (MIC) of carbon steel wasdirectly proportional to bacterial resistance to metal ions.

The technological advancement of this invention is novel for the complexto be used as antitumor agent. In addition, it will be appreciated thatthe various compounds for making the complex nano particle and method ofusing the complex nano particle such as antibacterial activity orantitumor activity can be made. Accordingly, the specification anddrawings are to be regarded in an illustrative rather than a restrictivesense.

What is claimed is:
 1. A process to make a nanoparticle complex toreduce a cell viability, comprising: mixing stoichiometric amounts of aselenius acid and olylamine in an ethyl alcohol, stirring the solutiontill a precipitate is formed; filtering and washing the precipitate byethyl alcohol; crystallizing the precipitate by diethyl ether to form aolylammonium hydrogen selenite reacting a sodium carbonate with a metalcarbonate forming a metal carbonate precipitate; refluxing two moles ofthe olylammonium hydrogen selenite to one mole of the metal carbonate toform a metal olylammonium hydrogen selenite complex; and mixing andgrounding a cyclodextrin and the metal olylammonium hydrogen selenitecomplex to form the nanoparticle complex of cyclodextrin-metalolylammonium hydrogen selenite complex of a specific size to be used asan anti-tumor agent.
 2. The process of claim 1, wherein the precipitateis recrystallized by diethyl ether.
 3. The process of claim 1, whereinolylammonium hydrogen selenites formed has a general formula RN⁺H₃HSeO₃.4. A process, comprising: reacting a selenius acid with a metalcarbonate forming a precipitate; washing the precipitate followed by afiltration; leaving a filtrate at room temperature for crystallization;washing a crystal formed with water; drying the crystals in air andforming metal complex as a metal hydrogen selenite dehydrate complex andmixing the metal olylammonium hydrogen selenite dehydrate complex with acyclodextrin oligosaccharide to form a nanoparticle as a metal basedcationic surfactant to be used as an anti-cancer drug.
 5. The process ofclaim 4, wherein metal hydrogen selenite dehydrate complex is a Cobalthydrogen selenite dehydrate complex.
 6. The process of claim 4, whereinmetal hydrogen selenite dehydrate complex is a Platinum hydrogenselenite dehydrate complex.
 7. The process of claim 4, wherein seleniusacid and Platinum carbonate is mixed in equimolar amounts to formPlatinum hydrogen selenite dehydrate complex.
 8. The process of claim 4,wherein selenius acid and Cobalt carbonate is mixed in 1:2 ratio toforma Cobalt hydrogen selenite dehydrate complex.
 9. The process ofclaim 4, wherein the filtrate is left for 2 days for crystallization forthe formation of a Cobalt hydrogen selenite dehydrate complex.
 10. Theprocess of claim 4, wherein the filtrate is left for 24 hours forcrystallization for the formation of a Platinum hydrogen selenitedehydrate complex.